Malfunctions in the human circadian (~24 hr) timing system and/or mutations in 'clock' genes are implicated in many disorders and diseases including affective disorders, chronic sleep problems, a range of metabolic syndromes and even susceptibility to cancer. Based on many lines of evidence it is now established that time-of-day specific changes in the phosphorylation state of one or more central clock proteins is the key state variable setting the pace of circadian clocks. In animals, PERIOD (PER) proteins are the clock components behaving as the primary 'phospho-timer.' The importance of PER phosphorylation to human health is highlighted by studies showing that mutations in either a phosphorylation site on human PER2 or a kinase that phosphorylates PER underlie several familial advanced sleep phase syndromes (FASPS). A critical function of PER proteins is that they participate with other core clock proteins to generate a multi-component biochemical oscillator based on transcriptional negative feedback loops that not only perpetuate daily cycles in the expression of clock genes but also drive rhythmic expression of about 10 percent of a cell's transcripts, which ultimately underlies many of the observed circadian rhythms. PER proteins play a critical role in cyclical gene expression by acting in a phase-specific manner as 'scaffolds' to recruit multi-subunit 'repressor' complexes to central clock transcription factors. Time-of-day specific changes in PER phosphorylation are critical for generating circadian gene expression by limiting when in a daily cycle PER engages in transcriptional repression by regulating its abundance, timing of nuclear entry, duration in the nucleus and possibly repressor potency. Many of the core clock proteins, such as the central transcription factors, are phosphorylated, although the functions of these phosphorylation events are not clear. The work proposed herein will add significant new insights into the biochemical basis underlying circadian machineries by using the genetically tractable model organism Drosophila melanogaster to: 1) identify phosphorylation sites on key clock proteins, and determine their biochemical and physiological roles, 2) identify the relevant kinases and determine their roles in the clockworks, and 3) isolate native clock complexes, identify constituent factors and determine their role(s) in circadian rhythm generation. A more comprehensive view of the phospho-networks operating in the clockworks and its inter-relationships with clock complex assembly/function will be attained. PUBLIC HEALTH RELEVANCE: Humans exhibit daily changes in physiology and behavior, such as our wake-sleep cycles, that are controlled by a network of specialized cells called circadian clocks. A key gear in the timing mechanism of these cellular clocks is a protein called PERIOD that when mutated can lead to severe sleep disorders and other health-related problems. This application will undertake a multi-faceted strategy to investigate how PERIOD and other clock proteins contribute to the ticking of the clock.